Design, synthesis, and molecular docking studies of novel pomalidomide-based PROTACs as potential anti-cancer agents targeting EGFRWT and EGFRT790M

Abstract A new class of EGFR PROTACs based on pomalidomide was developed, synthesised, and tested for their cytotoxic activity against a panel of human cancer cells. Compounds 15–21 were showed to be more effective against the four tested cell lines than erlotinib. In particular, compound 16 was found to be the most potent counterpart as it was 5.55, 4.34, 5.04, and 7.18 times more active than erlotinib against MCF-7, HepG-2, HCT-116, and A549 cells, respectively. Compound 15 was revealed to be more active than doxorubicin against the four tested cell lines. Furthermore, the most potent cytotoxic compounds were studied further for their kinase inhibitory effects against EGFRWT and EGFRT790M using HTRF test. Compound 16 showed to be the most effective against both kinds of EGFR, with IC50 values of 0.10 and 4.02 µM, respectively. Compound 16 could effectively degrade EGFR protein through ubiquitination (Dmax = 96%) at 72 h in the tested cells.


Introduction
The epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase protein that regulates cell proliferation, invasion, metastasis, and apoptosis in human epithelial cells by acting as a receptor for members of the EGF family. 1-3 EGFR gene amplification has been associated to several human malignancies, including oesophageal cancer, glioblastoma, anal cancers, malignancies of the epithelium of the head and neck, breast cancers, and lung cancers, particularly non-small-cell lung cancers (NSCLCs). [3][4][5][6] Mutations in the EGFR kinase adenosine triphosphate (ATP)binding domain is considered as the oncogenic driver in NSCLC, such as in-frame deletions of exon 19 and the L858R mutation. 1 Lung cancer is the most common cancer related to death in the world, and NSCLCs are one of the most common types of lung cancer. 7 The biomedical community has actively examined EGFR as a therapeutic target for NSCLC. Studies on inhibiting the activity of mutant EGFR ATP-binding domains resulted in the creation of a variety of FDA-approved EGFR tyrosine kinase inhibitors (TKIs). In NSCLC patients, the first-generation of TKIs, gefitinib 8 and erlotinib, 9 exhibited significant responses and prolonged survival rates. The secondary "gatekeeper" T790M mutation, on the other hand, enhanced ATP-binding affinity and induced recurrence in the majority of NSCLC patients after 9-14 months of treatment. [10][11][12][13][14] To prevent resistance, second-generation of EGFR inhibitors have been developed, including afatinib and dacomitinib, which target EGFR with the T790M activating mutation. Following that, the third-generation of EGFR covalent inhibitors was created, with increased selectivity to (WT) EGFR 13 . However, acquired resistance to irreversible EGFR-TKIs has been linked to the C797S point mutation and/or other mechanisms, making NSCLC resistant to these inhibitors. [15][16][17][18][19] The fourth-generation EGFR-TKIs, such as EAI045 20 and other noncovalent inhibitors targeting allosteric binding site (s), appears to be a substantial break through against these tertiary mutations. [21][22][23] Despite this progress, there is still a medical need for new small-molecule inhibitors or therapeutic methods to overcome multipoint EGFR mutations. 24 Proteolysis targeting chimaeras (PROTACs) can target a specific protein for degradation as a potential therapeutic method. [25][26][27][28][29][30] PROTAC-induced proximity causes preferential polyubiquitination of the target protein, which leads to proteasome destruction. Unlike typical enzyme inhibitors, which limit the target enzyme's catalytic activity, PROTACs cause the target protein to degrade. As a result, the new bifunctional small-molecule-mediated protein degradation paradigm has the potential to overcome the disadvantages of traditional occupancy-driven inhibitors. This method has been successfully used for the degradation of a variety of proteins in recent years 31 .
Meng et al. developed a set of putative EGFR degraders (EGFR PROTAC 1-3) in order to investigate a potential novel therapeutic strategy for NSCLC and overcome drug resistance ( Figure 1). As shown in (Figure 1), two new CRBN-based EGFR targeting PROTACs (SIAIS125 and SIAIS126) were reported to induce degradation for both EGFR Ex19del and EGFR L858R/T790M resistant proteins. It is noticeable from their chemical structures that they are based on pomalidomide. Additionally there are EGFR targeting small molecule PROTACs based also on pomalidomide ( Figure 1) that have showed selective and potent antitumor activities in EGFR-TKI resistant lung cancer cells and can stimulate necrosis and stop cell cycle in H1975 cells. 31 All these facts encouraged us to design a new pomalidomide based EGFR targeting PROTACs to get new hopeful candidates that can affect both types of EGFR either wild or mutant.
These findings imply that these compounds with EGFR inhibitory action could be useful in anticancer treatments. We used compounds 1-7 as ligands of EGFR to try to find a new EGFR degrader. The design, synthesis, and bioactivity evaluation of dioxopiperidinyl moiety derivatives 15-21 as new EGFR degraders were described in this study. The CRBN ligand pomalidomide and the VHL ligand were chosen as E3 ligase-recruiting components in our EGFR-targeting PROTACs (Figure 2(B)).
Target compounds 15, 16 and 17 were obtained (56-60% yields, purification by flash chromatography) through the reaction of compound 14 with quinoxalines 1-3 by heating under reflux at 80 C overnight, using methyl-2-pyrrolidone (NMP) as solvent and DIPEA as a base.
Synthesised compounds 18 and 19 (52-56% yield, purification by flash chromatography), were obtained by the same previous method via the treatment of intermediate key 14 with isatin hydrazides 4 and/or 5, respectively.
Furthermore, the reaction of compound 14 with benzoxazole hydrazide 6 and benzothiazole hydrazide 7, resulted in the formation of target compounds 20 and 21, respectively (50-52% yield, purification by flash chromatography) by the same previous method (Scheme 2). Proposed structures for EGFR PROTACs degraders 15-21 were in agreement with their various spectroscopic and analytical data (Supplementary data files).

Egfrwt kinase inhibitory assay
EGFR WT kinase inhibiting activities of the target compounds 15-21 were investigated using the homogeneous time resolved fluorescence (HTRF) assay, 44 with erlotinib as a standard (

Correlation of cytotoxicity with EGFR WT inhibition
The target compounds, 15-21 exhibited an inhibitory activity against EGFR WT . Next, we evaluated whether the EGFR WT inhibition can lead to an antiproliferative effect in the tested four cell lines. Using the Graph Pad-Prism 5 software, the activity of the tested compounds as EGFR WT inhibitors was plotted against their cytotoxicity in a simple linear regression configuration. The measured coefficients of determination (R 2 ) represent the relationship between EGFR WT inhibition and the induced antiproliferative activity. The

Western blotting assay
The western blotting analysis showed that, the majority of our target compounds 15-21 are moderate to good degraders ( Figure  4(A)). Degradation at the concentration of 1 mM is significantly higher than that at 0.1 mM, indicating a certain concentrationdependent relationship. Following this, the target compounds 15-21 tethering various E3 ligases ligand were chosen as representative PROTACs for further degradation study based on temporal data.
We set nine concentration gradients ranging from 1 mM to 10 mM to determine the degradation activity of all tested compounds and calculated the DC 50 (concentration that caused deletion of 50% of EGFR) values. The results showed that, compounds 15 and 16 can induce EGFR WT degradation in A549 cells in a concentration-dependent manner, with DC 50 values of 43.4 and 32.9 nM, respectively. At greater concentrations of compound 16, it exhibited a significant "hook effect" on EGFR degradation, which was caused by the creation of unproductive dimers (rather than productive ternary complex), 35 while compounds 17-21 were moderately effective at the concentrations of 1 mM to 10mM (Figure 4(B)).
At a concentration of 100 nM, the time-dependent degradation activities of compounds 15-21 were also examined ( Figure 5(A)). As the administration duration was extended, the amount of EGFR protein was gradually reduced. At 96 hours, compound 15 reached its maximum degradation rate (D max ¼ 86%) at 96 h, and compound 16 showed the maximum degradation rate (D max ¼ 96%) at 72 h. These results indicate that transmembrane protein degradation is a time-consuming process. Moreover, as shown in ( Figure  5(B)), compounds 15 and 16 at the concentrations of 0.1, 0.3 and 1 mM effectively displayed inhibitory activity against EGFR and downstream Akt phosphorylation in a concentration-dependent manner.
Compound 15 inhibited EGFR phosphorylation in a similar way as erlotinib, whereas compounds 15 and 16 have inhibitory actions on Akt phosphorylation that were comparable to erlotinib. In A549 cells, the promising compounds 15 and 16 promoted EGFR degradation with DC 50 values of 43.4 and 32.9 nM, respectively. Protein-controlling machinery in cells (ubiquitin-proteasome system) UPS was a part of the process.
2.2.6. In vitro DNA-flow cytometric (cell cycle) analysis Cell cycle analysis on MCF-7, HepG-2 and HCT-116 cells was performed for the most potent compound 16, as indicated by Wang et al. 45 Compound 16 was incubated with MCF-7, HepG-2, and HCT-116 cells for 24 hours at doses equivalent to its IC 50 against the three cell lines (3.92, 3.02 and 3.32 mM, respectively). Then, the influence of compound 16 on the cell cycle profile was then investigated.  When MCF-7 pre-treated to compound 16, the percentage of cells in pre-G1 and G2-M phases increased by 4.39 and 1.53 fold, respectively, compared to the control. In HepG-2 cells, compound 16 caused 6.76 and 1.61 fold increase in the percentage of cells in pre-G1 and G2-M stages, respectively, as compared to the control. In HCT-116 cells, compound 16 caused 5.37 and 1.49 fold increase in the percentage of cells in pre-G1 and G2-M phases, respectively, as compared to the control. These results clearly showed that compound 16 inhibits the cell cycle in the G2-M phase ( Figure 6).

Apoptosis analysis
The double staining Annexin V/propidium iodide technique was performed to assess the mechanism of cell death and apoptosisinducing activity. 46 For 24 hours, MCF-7, HepG-2, and HCT-116 cells were treated with compound 16 at concentrations of 3.92, 3.02 and 3.32 mM, respectively. As shown in (Figure 7 (A and B).
The results revealed that the application of compound 16 on MCF-7 cells the early apoptosis ratio jumped from 0.67% to 4.18%, and the late apoptosis ratio from 0.56% to 5.07%, compound 16 promoted nearly 9-times for cellular apoptosis, including early and late when compared to control. HepG-2 cells, compound 16 improved the early apoptosis ratio from 0.57% to 7.55%, while the late apoptosis ratio rises from 0.49% to 5.53%. This means that compound 16 caused almost up to 13-folds for both early and late cellular apoptosis upon comparison with the control. Compound 16 enhanced the early apoptosis ratio in HCT-116 cells from 0.63% to 5.96%, as well as the late apoptosis ratio from 0.53% to 5.15%, upon comparison with the control, compound 16 caused nearly 9-times for cellular apoptosis, including early and late. Compound 16 has a significant apoptotic effect against MCF-7, HepG-2, and HCT-116 cells, according to the obtained results.

Caspase-3 determination
The most sensitive cells (HepG-2) were treated with compound 16 at a concentration of 3.02 mM for 24 hours to determine the effect of compound 16 on caspase-3 levels, The results showed that considerable increase the level of caspase-3 (6.31 fold) when compared to control cells (Figure 7 (C)).
The commercial programme Molecular Operating Environment (MOE) 2019.01 was used to construct a molecular docking protocol. The structural coordinates of the co-crystallized inhibitors were used to determine the active binding sites of the target proteins. The docking protocol's results were verified by re-docking the co-crystallized ligands (erlotinib and TAK-285) inside the active sites of EGFR WT and EGFR T790M , respectively. The root mean square deviations (RMSDs) of erlotinib and TAK-285 re-docked conformers and co-crystallized conformers, respectively, were 1.5 and 0.90, demonstrating the docking process' validity ( Figures 8 (A), 10 (A)).
The binding energy of erlotinib as a co-crystallized ligand was À8.24 kcal mol À1 .The quinazoline moiety been located in the adenine pocket of EGFR WT , where the pyrimidine rings creating one hydrogen bonding with Met769 with a distance of 2.22 Å. The quinazoline molecule's phenyl ring was integrated into pi-Sigma linkages with Lue694 and Leu820. The terminal ethynylphenyl moiety was coupled with the hydrophobic pocket I, resulting, hydrophobic interactions with Ala719, Val702 and Lys721 residues. In the hydrophobic region II, two 2-methoxyethoxy groups were discovered generating hydrophobic interactions with Gly772 and Leu694 residues and one hydrogen bond with Cys773 (Figure 8 (B)).
The binding mode of target compound 15 was similar to that of erlotinib. It interacts with the active site through two hydrogen bonds with Asp831 and Lys721 amino acids with a distance of 3.09 and 3.14 Å, respectively and by hydrophobic interactions with Leu694 and Val702 amino acids with bond lengths of 4.32 and 4.60 Å, respectively [binding score ¼ À7.50 kcal mol À1 ] (Supplemental data).
Compound 16 interacted with the active site by two interactions, two hydrogen bonds with Arg779 and Lys721 amino acid with a distance of 3.07 and 2.79 Å, respectively, and by hydrophobic interactions with Lys889 amino acid with a distance of 3.72 and 4.34 Å, respectively [binding score ¼ À7.38 kcal mol À1 ] ( Figure 9).
Finally, the interactions between compound 17 and the active site are represented by four hydrogen bonds with Lys851, Lys721 and Phe699 amino acids with a distance of 3.14, 3.14, 2.98  3.14 Å respectively [binding score ¼ À7.68 kcal mol À1 ] (Supplemental data).
The docked compounds 15-21 have revealed good binding affinities against EGFR T790M (energy score À7.58 to À8.41 kcal mol À1 , Table 3). As a co-crystallized ligand, TAK-285's pyrrolo [3,2d]pyrimidine moiety occupied the adenine pocket of EGFR T790M (energy score À8.18 kcal mol-1). ALa734 and Leu718 have developed hydrophobic contacts with it. With a bond length of 2.23 Å, the pyrimidine moiety made one hydrogen bond with Met793. The terminal 3-(trifluoromethyl) phenoxy group was integrated into the hydrophobic pocket I, resulting in hydrophobic interactions at Lys745 and Ile759. With a bond length of 1.41 Å, the trifluoromethane group formed one hydrogen bond with Lys745. The N-ethyl-3-hydroxy-3-methylbutanamide moiety occupied the hydrophobic region II, forming one hydrogen bond with Ser720 with a length of 1.79 Å. With Lys745 Val726 and Met790, the central phenyl moiety formed pi-Sigma connections ( Figure 10).
Compound 15 bonded with two significant hydrogen bonds with Glu762 and Lys745 amino acids with a distance of 3.11 and 2.84 Å, respectively, and by hydrophobic interactions with Leu718 amino acid with a distance of 4.47 Å [binding score ¼ À7.95 kcal mol À1 ] (Supplemental data).
Compound 16 had a binding mechanism that was similar to TAK-285, it interacts with the active site through two hydrogen bonds with Ser720 and Lys745 amino acids with a distance of 3.28 and 3.36 Å, respectively, and by hydrophobic interactions with Leu718 amino acid with bond lengths of 4.54 Å [binding score ¼ À7.58 kcal mol À1 ] (Figure 11). Compound 17 showed a binding mode identical to TAK-285, it interacts with the active site through four hydrogen bonds with Asp837, Cys797, Leu718, and Met790 amino acids with a distance of 3.48, 3.39, 4.20, and 3.03 Å [binding score ¼ À7.73 kcal mol À1 ] (Supplemental data). The previously mentioned interactions indicate the importance of aromatic moieties, the H-acceptor and Hdonor in the designed ligands; this also maintains an appropriate lipophilicity of the designed compound to introduce appropriate pharmacokinetics.

General chemistry
All reagents in this study were provided from Merck, Aldrich, and Fluka. Thin-layer chromatography (TLC) was used to monitor all reactions, with precoated plates of silica gel G/UV-254 of 0.25 mm thickness (Merck 60F254) and UV light (254 nm/365 nm) for visibility. The attenuated total reflection (ATR) method was used to measure infra-red spectra with an FT-IR-ALPHBROKER-Platinum-ATR spectrometer and NMR spectra were performed on Bruker Spectrophotometer (400 MHz, 100 MHz for 1 H-13 C NMR, respectively). Chemical shifts are measured in d values parts per million (ppm) with comparison to the internal reference tetramethylsilane (TMS).
Coupling constants (J) for 1 H NMR were given in Hertz and expressed as (s) for singlet, (d) for doublet, (t) for triplet, (q) for the quartette, and (m) for multiplet, DMSO-d 6 was used as a solvent. Microanalyses (C, H, N, and S) and the results were within ± 0.4% of the theoretical values.

In vitro antiproliferative activities
Using the MTT assay technique, the antiproliferative properties of target compounds 15-21 were evaluated in vitro against MCF-7, HepG-2, HCT-116, and A549 cell lines. 39,40,50,51 The used cell lines were procured from ATCC (American Type Culture Collection). The anti-proliferative activity was quantified in the following way. At a density of 3-8 Â 10 3 cells per well, human cancer cell lines were introduced to 96-well plates. The wells were then incubated at 37 C for 12 hours in 5% CO 2 incubator. To determine the DMSO content, the culture media was swapped with 0.1 ml of fresh medium containing graded amounts of the test compounds for each well. Incubation time for the wells was two days. The cells were then cultured in 100 ml MTT solution (5 mg ml À1 ) for another 4 hours in each well.
The absorbance of each well was measured at 490 nm with an automated ELISA reader system (TECAN, CHE) after MTT-formazan crystals were dissolved in 100 ml of DMSO. The IC 50 values were calculated using nonlinear regression fitting models (Graph Pad, Prism Version 5). The results were expressed as means ± SD and were based on the average of three separate, duplicate trials.

Egfrwt and EGFR T790M kinase inhibitory assay
The inhibitory actions of target compounds 15-21 against both EGFR WT and EGFR T790M were investigated further after they demonstrated promising IC 50 values against target cell lines. This test used HTRF 44 assay with EGFR WT and EGFR T790M (Sigma). For the first 5 minutes, the evaluated compounds were incubated with EGFR WT and/or EGFR T790M and their substrates in the enzymatic buffer. ATP (1.65 mM) was allowed to react to start the enzymatic activity. The assay was performed at 37 C for 30 minutes. The addition of EDTA-containing detection reagents halted the process. After one hour detection period, GraphPad Prism 5.0 was used to calculate the IC 50 values. For each concentration, three separate trials were carried out.

Western bolt assay
Cells were seeded in 6-well plates at 1 Â 10 6 per well, incubated at 37 C with 5% CO 2 for 24 h before drug exposure. Cells were treated with different concentrations of synthesised compounds for 96 h, then collected and suspended in lysis buffer (Beyotime) and centrifuged for 20 min at 12000 rpm, later removed the insoluble material. The same amounts of proteins were loaded and separated by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore) after that. The results were detected by an enhanced chemiluminescence system (Mill-pore). The anti-EGFR was diluted at1:1000 (Cell Signalling Technology) were diluted at 1:2000. Anti-actin and the secondary antibodies (Xi'an Zhuangzhi Biotechnology Co., Ltd.) were diluted at 1:5000 and 1:10000, respectively.

Cell cycle analysis
MCF-7, HepG-2, and HCT-116 cells were given the most active compound 16 at concentrations of 3.92, 3.02 and 3.32 mM for 24 hours, respectively. The cells that had been tested were then trypsinized and washed in sterilised phosphate buffer saline (PBS). The collected cells were fixed with cold ethanol (100%, 1.5 ml). According to the manufacturer's instructions, a Cycle TESTTM PLUS DNA Reagent Kit was used to dye the cells (BD Biosciences, San Jose, CA). A flow cytometer was used to assess cell-cycle distribution. 52 3.2.5. Cell apoptosis assay MCF-7, HepG-2, and HCT-116 cells were seeded and grown overnight, then treated for 24 hours with compound 16 at concentrations of 3.92, 3.02, and 3.32 mM, respectively, to test if it caused apoptosis. The negative control was chosen to be DMSO. The cells were then collected and washed twice in PBS. The cells were separated using centrifugation. An apoptosis detection kit (BD Biosciences, SanJose, CA) was used in this investigation. Following the manufacturer's instructions, the cells were dyed with Annexin V-FITC and propidium iodide (PI) in the binding buffer for 20 minutes at 37 C in the darkness. A flow cytometer was used to assess the binding of Annexin V-FITC and PI. The frequencies in all quadrants were investigated using the Flowjo program. 53 3.2.6. Caspase-3 determination Caspase-3 activation was measured using a Caspase-3 ELISA Kit (KHO1091).

Conclusions
We have developed a new class of EGFR PROTACs degraders which were based on pomalidomide. All compounds were evaluated for antiproliferative effects in vitro and demonstrated potent inhibitory effect on MCF-7, HepG-2, HCT-116, and A549 cell lines. Compound 16 showed to be 5.55, 4.34, 5.04, and 7.18 folds more active than erlotinib in MCF-7, HepG-2, HCT-116, and A549 cells, respectively. For the target compounds 15-21, inhibitory properties against two isoforms, EGFR WT and EGFR T790M , were examined. The target compounds showed promising activities towards both wild-type and mutant forms. Compound 16 revealed to be the most effective EGFR inhibitor with IC 50 values of 0.10 and 4.02 mM against EGFR WT and EGFR T790M , respectively. To have a better understanding of the effect of the target compounds on cancer cell growth inhibition, docking experiments were conducted to fully visualise and interpret compounds' inhibitory profile against EGFR WT and EGFR T790M , and they revealed that the majority of the target compounds have comparable binding mechanisms with the target co-crystallized ligand. Therefore, this study presents compound 16 as a potential promising candidate as an EGFR inhibitor. The western blotting assays analysed the effect of EGFR degradation and the results showed the promising compounds 15 and 16 induced degradation of EGFR in A549 cells with the DC 50 values of 43.4 and 32.9 nM, respectively. Cellular protein-controlling machinery UPS was involved in this process, compound 16 could effectively degrade EGFR protein through ubiquitination and reached the maximum degradation rate (D max ¼ 96%) at 72 h.